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PubMed2005

Structural investigation of edible zein films/coatings and directly determining their thickness by FT-Raman spectroscopy.

Near-infrared Fourier transform Raman (FT-Raman) spectroscopy was employed to study the molecular structure of edible zein films/coatings, which were fabricated directly from zein protein. The secondary structure of zein protein was mainly in alpha-helix and remained unaltered during film formation as evidenced by the vibrational modes of amide I at 1656 cm(-1) and amide III at 1274 cm(-1). Raman results indicated that hydrophobic interaction played an important role in the formation of zein film and disulfide bonding might be responsible for the structural stability of zein protein during film formation. To enhance its antimicrobial property, an antimicrobial zein film was manufactured by incorporating zein protein with benzoic acid whose structure was then characterized by FT-Raman. It showed that physical entrapment or hydrophobic interaction was crucial to the incorporation of benzoic acid with zein protein, and the secondary structure of the antimicrobial film was still maintained in alpha-helical form. In addition, FT-Raman exhibits its preference in directly determining the thickness of zein films/coatings. By correlating the Raman intensity ratio of nu(1003) to nu(84) (I(1003/84)) versus the thickness of zein film, a linear relationship with high coefficient (R(2) = 0.9927) was obtained, which was then used pragmatically to determine the thickness of zein coatings on apple. It showed that the FT-Raman result (thickness = 0.27 +/- 0.01 mm) was consistent with that of classical micrometric measurement (thickness = 0.28 +/- 0.02 mm). Consequently, FT-Raman provides a direct, simple, and reagent-free method to characterize the structure and the thickness of zein films/coatings.

Anti-Infective Agents↗
PubMed2001

Genomics analysis of genes expressed in maize endosperm identifies novel seed proteins and clarifies patterns of zein gene expression.

We analyzed cDNA libraries from developing endosperm of the B73 maize inbred line to evaluate the expression of storage protein genes. This study showed that zeins are by far the most highly expressed genes in the endosperm, but we found an inverse relationship between the number of zein genes and the relative amount of specific mRNAs. Although alpha-zeins are encoded by large multigene families, only a few of these genes are transcribed at high or detectable levels. In contrast, relatively small gene families encode the gamma- and delta-zeins, and members of these gene families, especially the gamma-zeins, are highly expressed. Knowledge of expressed storage protein genes allowed the development of DNA and antibody probes that distinguish between closely related gene family members. Using in situ hybridization, we found differences in the temporal and spatial expression of the alpha-, gamma-, and delta-zein gene families, which provides evidence that gamma-zeins are synthesized throughout the endosperm before alpha- and delta-zeins. This observation is consistent with earlier studies that suggested that gamma-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD gamma-zein, an 18-kD alpha-globulin, and a legumin-related protein. Immunolocalization of the 50-kD gamma-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other gamma-zeins. The 18-kD alpha-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of two types of 22 kilodalton alpha-zein genes in a gene cluster in maize.

Five genes of the alpha-zein subfamily four (SF4) are located in a 56 kb genomic region of the maize inbred line W22. Their nucleotide and deduced amino acid sequences have been determined. The sequences define two types of alpha-zein SF4 genes-type 1 (T1) and type 2 (T2). The single T1 alpha-zein SF4 gene codes for an alpha-zein protein with a M(r) of about 22,000. This is the first alpha-zein SF4 gene sequenced that contains no early in-frame stop codons in its coding sequence. The four T2 alpha-zein SF4 genes in this cluster contain one or two early in-frame stop codons. In addition, our T1 and T2 genes differ markedly in the base sequences of their distal 5' non-translated flanking regions. The nucleotide and the deduced amino acid sequences of these two types of alpha-zein SF4 genes are similar (greater than 90%) to one another and to all known alpha-zein SF4 genes and cDNAs. Of the known W22 alpha-zein SF4 genes, only one in six does not contain an early in-frame stop codon. If the number of alpha-zein SF4 genes is 15-20, then we estimate that only about 4 of the W22 alpha-zein SF4 genes are without in-frame early stop codons.

Amino Acid Sequence↗
PubMed2003

Allelic variation and differential expression of methionine-rich delta-zeins in maize inbred lines B73 and W23a1.

The sulfur-amino-acid-rich delta-zeins of maize ( Zea mays L.) are represented by 18-kDa and 10-kDa proteins. We have cloned a novel 11-kDa methionine-rich delta-zein from developing endosperm of the inbred line W23a1. The nucleotide sequence of this new delta-zein is identical to the published 10-kDa delta-zein, except for an insertion of 18 nucleotides between +316 and +333 bp from the translation start site. Antibodies raised against the recombinant 18-kDa delta-zein recognized both the 18-kDa and 10-kDa delta-zein from total seed protein extracts of different maize inbred lines. Western blot analysis revealed differences in the levels of the delta-zeins in different inbred lines and some of the inbred lines lacked either the 10-kDa or the 18-kDa delta-zeins. Northern blot analysis revealed temporal differences in the RNA transcript levels of the 11-kDa and 18-kDa delta-zeins between B73 and W23a1. Such differences were not evident on Western blot analysis where similar protein accumulation profiles were seen for both lines. Immunostaining of paraffin sections of developing maize endosperm with the 18-kDa delta-zein antibodies revealed specific labeling of protein bodies found in the first few starchy layers from the aleurone layer. Electron-microscopic observation of thin-sections of B73 and W23a1 endosperm cells confirmed the presence of recently discovered novel, vacuole-like structures in these inbred lines. Immunogold labeling studies revealed that the delta-zeins were localized in the endoplasmic-reticulum-derived protein bodies and showed no preferential gold particle labeling over either the light or electron-dense material found in these protein bodies.

Alleles↗
PubMed2004

Effects of heating on the interaction of lipid and zein in a dry powder system.

The effects of heat treatment on the interaction of lipid and zein in a dry powder system were investigated. Linolenic acid ethyl ester (LAE) was mixed with the zein powder. The glass transition temperature (T(g)) for the dry powder zein was shown to be approximately 107 degrees C by differential scanning calorimetry. The thermogram of the zein-LAE mixed powder showed an exothermic transition near the T(g) of zein. Fourier transform infrared (FT-IR) spectroscopy was used for detecting the structural changes in zein by heat treatment, that is, elevating the temperature from 25 to 160 degrees C. The heat treatment of the powdery zein with and without LAE caused increases in the alpha-helix, beta-turn, and beta-sheet, concomitant with decreases in the intermolecular hydrogen-bonded beta-sheet and random coil. Such changes in the secondary structure were more drastic for the powder with LAE. The heating of the zein-LAE mixed powder also caused decreases in the peaks originating from LAE in the FT-IR spectra. These results suggest that the heat treatment induced the interaction of the zein and LAE in the powdery system. The influence of heating on the antioxidative activity of dry powder zein was studied by measurements of the peroxide value. When zein-LAE mixed powder was heated before storage, the oxidation of LAE was inhibited for 7 days, whereas LAE was oxidized within 1 day in the absence of heat treatment.

Drug Interactions↗
PubMed2004

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of zeins in mature maize kernels.

A high-throughput method has been developed to allow rapid analysis of maize seed storage proteins by matrix-assisted laser desorption time-of-flight mass spectrometry. The extraction solution containing an organic solvent, a reducing agent, and a volatile base has been optimized to enable extraction of all classes of zein proteins (alpha-, beta-, gamma-, and delta-). A near-saturating concentration of matrix, 2-(4-hydroxyphenylazo)benzoic acid, was necessary to obtain strong peaks for the most lipophilic zeins, the alpha-zeins. Zein proteins with small mass differences, difficult to separate by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were resolved through this analysis. Mass signals corresponding to the 10-kDa delta-, 15-kDa beta-, 16-kDa gamma-, 27-kDa gamma-, and several 19 and 22-kDa alpha-zeins were detected. The zein identities were further confirmed by the association of the number of cysteine residues in each zein MS peak, as determined by iodoacetamide derivatization, with the number predicted from its coding sequence. The relative zein abundance in the zein MS peaks was also correlated with the relative zein EST abundance among endosperm EST libraries. This method was utilized to examine the zein composition of a number of corn inbred lines and opaque mutants.

Hydrogen-Ion Concentration↗
PubMed2006

Antioxidant activity of zein hydrolysates in a liposome system and the possible mode of action.

Maize zein was hydrolyzed for 0.5-5 h by alcalase or papain. Protein solubility increased (P < 0.05) with the degree of hydrolysis (DH) and was higher for alcalase-hydrolyzed zein than for papain-hydrolyzed zein. The zein hydrolysates with both enzymes consisted mostly of small peptides or amino acids nondetectable by 15% acrylamide gel electrophoresis. Alcalase-hydrolyzed zein exhibited a stronger (P < 0.05) antioxidant activity than papain-hydrolyzed zein, as indicated by peroxide and thiobarbituric acid-reactive substance values in a liposome-oxidizing system. Zein hydrolysates possessed strong Cu(2+) chelation ability and marked reducing power, both of which were accentuated with hydrolysis time. The protein hydrolysates also showed strong radical-scavenging ability, which was not influenced by hydrolysis time. The antioxidant activity of alcalase-hydrolyzed zein at some specific low concentrations was close or comparable to those of butylated hydroxyanisole, alpha-tocopherol, and ascorbate. Although intact zein displayed an antioxidative effect, it was far less potent than hydrolyzed zein. The results demonstrated that enzyme-hydrolyzed zein can act as a metal ion chelator or a hydrogen donor, as well as a radical stabilizer to inhibit lipid oxidation. The effectiveness of the protein hydrolysates appeared to depend on both the concentration and the peptide/amino acid composition of the soluble protein fraction.

Amino Acids↗
PubMed1997

Lysine-rich modified gamma-zeins accumulate in protein bodies of transiently transformed maize endosperms.

During maize seed development, endosperm cells synthesize large amounts of storage proteins, alpha-, beta-, and gamma-zeins, which accumulate within endoplasmic reticulum (ER)-derived protein bodies. The absence of lysine in all zein polypeptides results in an imbalance in the amino acid composition of maize seeds. We modified the maize gamma-zein gene through the introduction of lysine-rich (Pro-Lys)n coding sequences at different sites of the gamma-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich gamma-zein constructs under the control of the 1.7 kb gamma-zein seed-specific promoter and the cauliflower mosaic virus (CaMV) 35S promoter. When (Pro-Lys)n sequences were inserted contiguous to or in substitution of the Pro-Xaa region of the gamma-zein, high levels of protein were observed. In contrast, when (Pro-Lys)n sequences were inserted five residues from the C-terminal, the transcript was present but modified protein was not detected. These results suggest that only an appropriate positioning of Lys-rich inserts leads to the modified molecule displaying correct folding and stability. Subcellular localization analyses and immunoelectron microscopy studies on isolated protein bodies demonstrated that modified gamma-zeins accumulate within these organelles and co-localized with endogenous alpha- and gamma-zeins. The studies reported here show the feasibility of manipulating the gamma-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.

Cloning, Molecular↗
PubMed2005

Cytoplasm and chloroplasts are not suitable subcellular locations for beta-zein accumulation in transgenic plants.

Zeins, the main storage proteins of maize that accumulate in the endoplasmic reticulum of the endosperm cells, are particularly interesting because they are rich in the essential sulphur amino acids. Overexpression of certain zein genes in plants such as alfalfa would be expected to improve the nutritional characteristics of this crop. Recently, significant accumulation values have been reached, but still far from those considered useful for nutritional purposes. This study investigates whether targeting to compartments other than the endoplasmic reticulum (cytosol and chloroplasts) could result in increasing beta-zein accumulation in transgenic plants. To address beta-zein to the cytosol, the fragment which codes for the signal peptide has been removed. beta-zein has also been targeted to alfalfa and tobacco chloroplasts by a transit peptide signal. Both tobacco, as a model plant species, and alfalfa have been transformed with the assembled constructs. An alternative route to accumulate beta-zein in the chloroplasts is to synthesize beta-zein directly in the plastid lumen. Thus, the beta-zein gene has also been inserted into tobacco plastid DNA. The beta-zein gene in each different type of transformed plant was properly transcribed, as determined by northern blot analysis, but no accumulation of beta-zein was detected, either in the cytoplasm or in the chloroplasts of alfalfa and tobacco transformed plants. Therefore, it is concluded that chloroplasts and the cytosol are not favourable subcellular locations for zein protein accumulation.

Animals↗
PubMed1991

Normal and lysine-containing zeins are unstable in transgenic tobacco seeds.

Chimeric genes composed of the beta-phaseolin promoter, an alpha-zein coding sequence and its modified versions containing lysine codons, and a beta-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. alpha-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa alpha-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an alpha-zein content that was approximately 0.003% of the total seed protein. The amount of alpha-zein in other transgenic plants varied between 1 x 10(-4)% and 1 x 10(-5)% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the alpha-zein protein. Polysomes translating alpha-zein mRNA isolated from tobacco seeds contained fever ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized alpha-zein was degraded in tobacco seeds with a half-life of less than 1 hour.

Lysine↗
PubMed2003

Protein-lipid interactions in zein films investigated by surface plasmon resonance.

Experiments on the adsorption of alpha-zein (characterized by SDS-PAGE) from aqueous ethanol and 2-propanol solutions onto hydrophilic and hydrophobic surfaces are reported. Zein adsorption onto self-assembled monolayers (SAMs) was detected by surface plasmon resonance (SPR). Gold substrates were prepared by thermal evaporation on glass slides. Gold-coated surfaces were modified by depositing SAMs of either a long-chain carboxylic acid terminated thiol [COOH(CH2)(10)SH] or a methyl-terminated alkanethiol [CH3(CH2)(7)SH]. Experimental measurements indicated that zein interacted with both hydrophilic and hydrophobic surfaces. Zein concentration affected the thickness of bound zein layers. The estimated thickness of the zein monolayer deposited on hydrophilic surfaces was 4.7 nm. Zein monolayer thickness on hydrophobic surfaces was estimated at 4.6 nm. The topography of zein layers was examined by atomic force microscopy (AFM) after solvent was evaporated. Surface features of zein deposits depended on the adsorbing surface. On hydrophilic surfaces, roughness values were high and distinct ring-shaped structures were observed. On hydrophobic surfaces, zein formed a uniform and featureless coverage.

2-Propanol↗
PubMed1995

Evidence against a potential endoplasmic reticulum transmembrane domain of 27K zein expressed in Xenopus oocytes.

During the cellular protein targeting process, zeins (maize storage proteins) are retained in the endoplasmic reticulum (ER) where they accumulate into protein bodies. There are circumstantial and preliminary data indicating that the 27K zein, a class of zein proteins, may span the ER membrane. This potential transmembrane domain is considered very significant with regard to the mechanism of ER retention for zeins. The potential transmembrane domain may permit the 27K zein to remain in the ER and to serve as an anchor for other classes of zein, thus acting as a nucleating factor for protein body formation. This study investigated the potential transmembrane feature in a heterologous system (Xenopus laevis oocyte). Following injection of synthetic 27K zein mRNA, isolated protein vesicles were subjected to proteinase K digestion, surface biotinylation and alkaline extraction. Throughout three categories of assay the possible role of the 27K zein as a transmembrane protein was consistently refuted in this study. The 27K zein polypeptide was affected by neither proteinase K digestion nor biotinylation and was released from alkali-stripped membranes. This study, therefore, concludes that the 27K zein is not a protein body nucleating factor by virtue of an ER transmembrane feature.

Animals↗
PubMed1997

A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane.

The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD alpha-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD alpha-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this alpha-zein reveals a putative mutation alanine-->valine (Ala-->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 alpha-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 alpha-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 alpha-zein and the fl2 alpha-zein (Val-->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 alpha-zein (Val-->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 alpha-zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.

Animals↗
PubMed1989

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.

Immunohistochemistry↗
PubMed1991

opaque-2 modifiers increase gamma-zein synthesis and alter its spatial distribution in maize endosperm.

Through the action of opaque-2 modifier genes, the soft, floury endosperm of opaque-2 mutants is converted to a vitreous phenotype. This change in endosperm texture is associated with a twofold to threefold increase in gamma-zein content. To investigate the effect of opaque-2 modifiers on the expression of gamma-zein genes, we analyzed the synthesis and distribution of gamma-zein protein and the level of gamma-zein mRNAs in developing endosperms of the inbreds W64A and W64Ao2, a modified opaque-2 mutant Pool 34 QPM, and their reciprocal F1 hybrids. We also characterized the number and organization of gamma-zein genes in these and related maize genotypes. Our studies show that opaque-2 modifiers are semidominant genes, resulting in a twofold to threefold increase in gamma-zein gene expression in both opaque-2 and normal genetic backgrounds. The increase in gene expression appears to be a consequence of enhanced mRNA transcription or stability rather than gene amplification because gamma-zein genes occur in one or two copies in modified as well as nonmodified genetic backgrounds. Ultrastructural studies showed that gamma-zein occurs in high concentrations in the first few subaleurone cells of nonmodified endosperms, but high concentrations of gamma-zein occur in the subaleurone and central endosperm cells of modified opaque-2 mutants. The increased concentration and distribution of gamma-zein in modified endosperms are highly correlated with the activity of opaque-2 modifier genes.

Microscopy, Immunoelectron↗
PubMed1984

Effect of RNase on Zein Synthesis in Endosperms of brittle-2opaque-2 Maize Double Mutant.

Posttranscriptional degradation of zein mRNAs in Zea mays, presumably caused by high RNase activity, may be involved in reducing zein synthesis in the endosperms of zein-deficient mutants. Zein proteins prepared from mature endosperms of zein-deficient double mutants such as brittle-2;opaque-2 showed an almost complete blockage of the synthesis of two major zein components, Z1 and Z2. However, these two components were present prior to 12 days postpollination. The changes in quantity and quality of zein synthesis in these double mutants correlated well with the rising RNase levels in the developing endosperm. In addition to its effect on zein synthesis, high RNase activity also influenced the stability of rRNAs in the brittle-2;opaque-2 double mutant at the later stages of endosperm development. These results suggest that RNase played a role in regulating zein synthesis in developing zein-deficient mutant endosperms.

Journal Article↗
PubMed2004

Expression of an 11 kDa methionine-rich delta-zein in transgenic soybean results in the formation of two types of novel protein bodies in transitional cells situated between the vascular tissue and storage parenchyma cells.

Soybean (Glycine max (L.) Merr.) is an important protein source in human diets and animal feeds. The sulphur content of soybean seed proteins, however, is not optimal for ration formulations. Thus, increasing the methionine and cysteine content of soybean seed proteins would enhance the nutritional quality of this widely utilized legume. We have earlier reported the isolation of an 11 kDa delta-zein protein rich in methionine from the endosperm of the maize (Zea mays L.) inbred line W23a1 [Kim, W.-S. and Krishnan, H.B. (2003) Allelic variation and differential expression of methionine-rich-delta-zeins in maize inbred lines B73 and W23a1. Planta, 217, 66-74]. Using Agrobacterium-mediated transformation, a construct consisting of the coding region of the cloned delta-zein gene under regulation of the beta-conglycinin alpha'-promoter was introduced into the soybean genome. The 11 kDa delta-zein gene was expressed in the seeds of transgenic soybeans, although low-level expression was also detected in the leaves. In situ hybridization indicated that the 11 kDa delta-zein mRNA was expressed predominantly in transitional cells located between the vascular tissue and storage parenchyma cells. Immunohistochemistry of developing transgenic soybeans revealed that the accumulation of the 11 kDa delta-zein occurred primarily in these transitional cells. Expression of the 11 kDa delta-zein gene in transgenic soybean resulted in the formation of two endoplasmic reticulum-derived protein bodies that were designated as either spherical or complex. Immunocytochemical localization demonstrated that both the spherical and complex protein bodies accumulated the 11 kDa delta-zein. Although expression of the 11 kDa delta-zein gene elevated the methionine content of the alcohol-soluble protein fraction 1.5-1.7-fold above that of the non-transgenic line, the overall methionine content of seed flour was not increased. Our results suggest that the confined expression of the 11 kDa delta-zein gene in transitional cells could be limiting the increase in methionine content in transgenic soybean seeds.

Journal Article↗
PubMed1984

Zein gene organization in maize and related grasses.

Zein cDNA clones were used to study the organization of zein genes within the genome of the inbred maize W64A. When individual clones for the two larger molecular-weight classes of zein proteins (Mr = 22,000; Mr = 19,000) were used as probes for Southern blot hybridizations of genomic DNA, multiple restriction fragments were found to hybridize. Reconstruction analyses using moderately stringent criteria were used to estimate a total of 70-80 zein sequences within the genome of this inbred maize. The hybridization patterns suggest that zein sequences are clustered within the same restriction fragment. When criteria permitting less cross-hybridization of homologous sequences (Tm - 10 degrees C) were used, the banding pattern changed, with some of the bands being reduced in intensity or eliminated entirely. Therefore, by control of hybridization criteria, particular zein genes may be more readily distinguished in a Southern blot analysis. The Southern blot hybridization pattern for the Mr = 15,000 zein was less complex. Only a single major band was found, with sufficient hybridization intensity for two or three genes. Genomic Southern analyses of other inbred maizes and related grasses showed similarly complex hybridization patterns with cDNA probes for the 19,000- and 22,000-molecular-weight zeins, suggesting that these sequences have been conserved over evolutionary time. The zein multigene family may therefore have arisen by gene duplication before divergence of the maize, teosinte, and Tripsacum species from a common ancestor.

Cloning, Molecular↗